Plant pests are a major factor in the loss of the world's important agricultural crops. Species of corn rootworm are considered the most destructive corn pests. Important rootworm pest species include Diabrotica virgifera virgifera, the western corn rootworm; D. longicornis barberi, the northern corn rootworm, D. undecimpunctata howardi, the southern corn rootworm, and D. virgifera zeae, the Mexican corn rootworm.
Corn rootworm is mainly controlled by intensive applications of chemical pesticides. Good corn rootworm control can thus be reached, but these chemicals can sometimes also affect beneficial organisms. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. This has been partially alleviated by various resistance management practices, but there is an increasing need for alternative pest control strategies. One such alternative includes the expression of foreign genes encoding insecticidal proteins in transgenic plants. This approach has provided an efficient means of protection against selected insect pests, and transgenic plants expressing insecticidal toxins have been commercialized, allowing farmers to reduce applications of chemical insecticides.
Bacillus thuringiensis (Bt) Cry proteins (also called 5-endotoxins) are proteins that form a crystalline matrix in Bacillus that are known to possess insecticidal activity when ingested by certain insects. Genes coding for Cry proteins have been isolated and their expression in crop plants have been shown to provide another tool for the control of economically important insect pests. Such transgenic plants expressing the Cry proteins have been commercialized, allowing farmers to reduce or augment applications of chemical insect control agents. Coleopteran-active Cry proteins useful in transgenic plants include, for example, Cry3A, Cry3B and the Cry34/Cry35 complex.
Although the usage of transgenic plants expressing Cry proteins is another tool in the insect control toolbox, it is still susceptible to resistance breakdown. Insect pests that now have resistance against the Cry proteins expressed in certain transgenic plants are known. A strategy to reduce the chances of resistance breakdown is to “stack” transgenic traits with different modes of action against the same insect pest species in a single plant. Currently, transgenic traits are frequently stacked through breeding and subsequent screening to get multiple transgenic traits in a single commercial germplasm. These breeding and screening steps are required for every variety of germplasm into which introduction of these two traits is desirable. For many agronomically important crops, such as corn, these two traits need to be maintained as hybrids for dozens of germplasm varieties. Additionally, factors such as the genetic linkage of undesirable traits or genetic recombination may complicate the introduction of two traits from two distinct loci into a single germplasm variety. Therefore, it would be advantageous to create a nucleic acid molecule which carries multiple insecticidal traits and can be introduced at a single locus in the genome of the transgenic plant.